Numerous GTP binding proteins (G proteins) and receptors that work by activating G proteins have been cloned in recent years. As this field matures an emerging pivotal problem in molecular pharmacology will involve determining the nature of the factors that control the interaction between receptors and G proteins. In order to begin to understand this interaction, we plan to examine, as a model system, the coupling between A1 adenosine receptors and G proteins. A remarkable feature of A1 receptors is their property of Interacting with several different effector systems in various tissues. Hypothesis 1 Is that the same A1 receptors are capable of interacting with several different G-proteins. We will use immunoblots to identity G proteins that are copurified by agonist affinity chromatography with A1 receptors from brain and adipose tissue. As a second approach, we will attempt to produce antibodies (by various means) that can be used to immunoprecipitate receptor-G protein (R-G) complexes. We have identified GH4 pituitary cells as a tissue culture line that has three different effector responses to A1 receptor activation (adenylyl cyclase, K+ channel conductance, and phospholipase C). Hypothesis 2a is that A1 receptors and various G proteins of GH4 cells become uncoupled at different rates during receptor desensitization. Hypothesis 2b is that coupling of various G proteins to A1 receptors of GH4 cells is differentially sensitive to pertussis toxin. To learn which G proteins produce the various A1 -receptor mediated effector responses, the time courses of: i) changes in adenosine-mediated effector responses; ii) the amounts of various G-proteins coupled to A1 receptors; and iii) other receptor and G protein characteristics; will be measured during desensitization and following exposure to graded doses of pertussis toxin. R-G complexes will be purified by agonist affinity chromatography or immunoprecipitation. As a third approach we will produce stable transfectants of GH4 cells that express high levels of A1 receptors that have been engineered to contain a polyhistidine residue at the carboxyl terminus. Polyhistidine containing proteins can be readily purified by Ni++ -NTA affinity chromatography. Hypothesis 3a is that myristylation of G protein alpha-subunits and the nature of G protein beta and gamma subunits influences coupling of G proteins to A1 adenosine receptors. Hypothesis 3b is that A1 receptor subtypes couple to various G proteins with differential selectivities. Reconstitution of receptor-G protein coupling will be assessed using homogeneous recombinant G-proteins expressed in insect cells, and homogeneous native or polyhistidine -containing A receptors. We anticipate that some of these approaches will be generalizable for use with other receptors that couple to G proteins.